Muscarinic-receptor-mediated changes in intracellular Ca2+ and inositol 1,4,5-trisphosphate mass in a human neuroblastoma cell line, SH-SY5Y.
This study reports increased intracellular Ca2+ and inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] in response to muscarinic-cholinergic stimulation of human neuroblastoma (SH-SY5Y) cells. Carbachol stimulation leads to a rapid increase in intracellular Ca2+ and Ins(1,4,5)P3 mass, both reaching a peak at around 10 s and then declining to a new maintained phase significantly above basal. Dose-response analysis of peak and plateau phases of intracellular Ca2+ shows different agonist potencies for both phases, carbachol being more potent for the plateau phase. The plateau-phase intracellular Ca2+ was dependent on extracellular Ca2+, which is admitted to the cell through a non-voltage-sensitive Ni2(+)-blockable Ca2+ channel. Using a Mn2+ quench protocol, we have shown that Ca2+ entry occurs early during the discharge of the internal stores. The plateau phase (Ca2(+)-channel opening) is dependent on the continued presence of agonist, since addition of atropine closes the Ca2+ channel and intracellular Ca2+ declines rapidly back to basal. We also failed to detect a refilling transient when we added back Ca2+ after intracellular Ca2+ had reached a peak and then declined in Ca2(+)-free conditions. These data strongly suggest that muscarinic stimulation of SH-SY5Y cells leads to a rapid release of Ca2+ from an Ins(1,4,5)P3-sensitive internal store and a parallel early entry of Ca2+ across the plasma membrane.
Lambert
D G
DG
University of Leicester, Department of Pharmacology and Therapeutics, U.K.
Nahorski
S R
SR
D001115:Arecoline / Q000494:pharmacology*; D001285:Atropine / Q000494:pharmacology; D001572:Benzofurans; D002118:Calcium / Q000378:metabolism*; D002217:Carbachol / Q000494:pharmacology*; D002460:Cell Line; D005456:Fluorescent Dyes; D016257:Fura-2; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism*; D015759:Ionomycin / Q000494:pharmacology; D007700:Kinetics; D008345:Manganese / Q000494:pharmacology; D009116:Muscarine / Q000494:pharmacology*; D009447:Neuroblastoma; D011976:Receptors, Muscarinic / Q000187:drug effects / Q000502:physiology*; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000378:metabolism*
D001572:Benzofurans; D005456:Fluorescent Dyes; D011976:Receptors, Muscarinic; D008345:Manganese; D001115:Arecoline; D015759:Ionomycin; D001285:Atropine; D009116:Muscarine; D015544:Inositol 1,4,5-Trisphosphate; D002217:Carbachol; D002118:Calcium; D016257:Fura-2